DESCRIPTION: The success of chemotherapeutics depends on the selective, efficient targeting of enzymes known to be dysregulated in specific cancers. Recently, in the treatment of multiple myeloma there has been increased interest in molecular targets from the ubiquitin proteasome system (UPS) including deubiquitinating enzymes (DUBs) due the clinical success of the proteasome inhibitors Bortezomib and Carfilzomib. DUBs are an essential component of the UPS and are responsible for regulating cellular protein levels and activating essential biological pathways such as protein degradation, cell division, and cell death. Furthermore, DUBs have recently been discovered to help promote Bortezomib resistance in multiple myeloma patients. However, there are no clinically available biochemical assays that directly measure DUB activity in primary tumor samples before and after treatment. Such an assay would be beneficial to (1) determine if patients would benefit from a DUB-targeted therapy, (2) identify an ideal dose of chemotherapeutic to maximize drug efficacy while minimizing side effects, and (3) analyze a heterogeneous sample to identify distinct subpopulations of drug-resistant cells. Current techniques attempt to recapitulate DUB activity in vitro; however it has become clear that these cell-free, test tube-based reactions do not adequately mimic in vivo conditions, resulting in the high failure rate of DUB-based therapeutics in phase 1 clinical trials Furthermore, these current methods often require large samples sizes and complex genetic engineering to indirectly measure enzyme activity. An interdisciplinary approach will be applied to develop a new method to quantify DUB activity in ex vivo samples that allows for direct measurement in intact single cells in a high-throughput manner. Unique fluorescent DUB reporters will be developed that are membrane-permeant, stable inside of the cell, and compatible with primary tumor samples without the need for complex genetic manipulations. The fluorescent biosensor will consist of the C-terminal region of ubiquitin as a substrate for DUB-mediated cleavage coupled with an N-terminal -hairpin `protectide' to increase the reporter lifetime in intact cells. This bioanalytical tool will then be incorporated into a droplet microfluidic device to allow for high-throughput screening (HTS) of an entire heterogeneous population of cells. Microfluidic devices are an ideal technology for HTS of cells due to their low reagent and materials cost, high reproducibility, and ease-of-use. The high-throughput single cell analysis will account for the significant heterogeneity associated with tumor biopsies and allows clinicians to identify distinct subpopulations of cells, including low-frequency, drug-resistant cells. The work proposed here is a technological first step towards a new analytical detection method to assist clinicians in the diagnosis and treatment of multiple myeloma.